Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses

Kouokam, Joseph Calvin; Lasnik, Amanda B.; Palmer, Kenneth E.

Fourteen daily doses of 10 mg/kg GRFT were injected s.c. to mice with a control group receiving the PBS vehicle only. All mice survived this treatment and animal behavior was similar in both GRFT and PBS treatment groups. Throughout the experiment, fitness (as determined by the animal body weight) was unchanged after treatment with GRFT ( Figure 4C ). In addition, there was no change in lung, kidney, liver, and heart weights after GRFT treatment (data not shown). However, splenomegaly was observed after sacrifice at Day 14: spleen/body weight ratios of 0.64% ± 0.15% and 0.37% ± 0.04% for GRFT and control groups, respectively ( Figure 4F ). Interestingly, splenomegaly was decreased considerably after two days of recovery, with spleen-to-body weight ratios of 0.52% ± 0.07% for animals treated with GRFT (vs. 0.4% ± 0.04% in the PBS group, p = 0.0546). Seven days post treatment, the difference in spleen/body ratios was not significant (0.41% ± 0.03% and 0.37% ± 0.04% for GRFT and PBS groups, respectively, p = 0.2874) as shown in Figure 4F . Of note, histological studies of kidneys and livers collected at Days 14 and 21 revealed no observable pathology induced by GRFT (data not shown). Despite the splenomegaly, we observed no adverse pathology due to GRFT treatment in spleen tissues after H and E staining. Representative spleens are shown in Figure 5A -C. Interestingly, segmental marginal zone lymphoid depletions were occasionally observed across all groups regardless of treatment. In order to study whether splenomegaly was due to spleen B-and T-lymphocyte activation, mice were treated for 14 days with 10 mg/kg GRFT. Relative to PBS-treated animals, we found that B220-only positive splenocytes were markedly increased in the GRFT group at Day 10 (210.21% ± 73.68% relative to PBS cells, p = 0.0151), a difference that vanished after a week of recovery ( Figure 5D ). Interestingly, there was no difference between relative amounts of all cells staining positive for B220 (CD19+/B220+ and B220+ only) in splenocytes obtained from PBS or GRFT animals at either time point ( Figure 5D ). As shown in Figure 5E , cells expressing the T-cell activation marker CD69 were slightly more abundant in animals treated with GRFT (148.10% ± 25.41% relative to the PBS group, p = 0.0221) at Day 10. By Day 17, the difference was markedly reduced with 116.24% ± 3.03% relative to PBS treated mice (p = 0.0295). Likewise, spleen cells expressing both CD4 and CD69 were slightly increased after 14 daily treatments with GRFT (137.28% ± 12.80% relative to PBS, p = 0.0043); this difference was reduced during recovery (121.95% ± 5.58% CD4+/CD69+ relative to PBS, p = 0.0488).